Six Cloned Calves Produced from Adult Fibroblast Cells after Long-Term Culture

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 97; no. 3; pp. 990 - 995
• Main Authors: Kubota, Chikara, Yamakuchi, Hiroshi, Todoroki, Junichi, Mizoshita, Kazunori, Tabara, Norio, Barber, Michele, Yang, Xiangzhong
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 01-02-2000; National Acad Sciences; National Academy of Sciences; The National Academy of Sciences
• Series: From the Cover
• Subjects: Animals; Biological Sciences; Cattle; Cattle - embryology; Cattle - genetics; Cell culture techniques; Cell Cycle; Cell Differentiation; Cells; Cells, Cultured; Cellular Senescence; Clones; Cloning, Organism; Cultured cells; Ear, External - cytology; Embryo Transfer; Embryonic cells; Epithelial cells; Female; Fiber cells; Fibroblasts; Fibroblasts - cytology; Genetics; Karyotyping; Male; Microsatellite Repeats; Oocytes; Pregnancy; Somatic cells; Starvation; Telomere - physiology; Totipotent stem cells
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.97.3.990

Cloning whole animals with somatic cells as parents offers the possibility of targeted genetic manipulations in vitro such as "gene knock-out" by homologous recombination. However, such manipulation requires prolonged culture of nuclear donor cells. Previous successes in cloning have been limited to the use of cells collected either fresh or after short-term culture. Therefore, demonstration of genetic totipotency of cells after prolonged culture is pivotal to combining site-specific genetic manipulations and cloning. Here we report birth of six clones of an aged (17-year-old) Japanese Black Beef bull using ear skin fibroblast cells as nuclear donor cells after up to 3 months of in vitro culture (10-15 passages). We observed higher developmental rates for embryos derived from later passages (10 and 15) as compared with those embryos from an early passage (passage 5). The four surviving clones are now 10-12 months of age and appear normal, similar to their naturally reproduced peers. These data show that fibroblasts of aged animals remain competent for cloning, and prolonged culture does not affect the cloning competence of adult somatic donor cells.